Inhibition Of CD69 For Treatment Of Inflammatory Conditions

ABSTRACT

A method of treating or reducing at least one inflammatory condition or the susceptibility to at least one inflammatory condition is provided involving administering at least one CD69 antagonist to a subject, wherein the subject has been diagnosed with at least one inflammatory condition, or a susceptibility to the same. CD69 antagonists can include one or more of an anti-CD69 antibody, an anti-CD69 aptamer, a CD69 mRNA antagonist, and a small molecule pharmaceutical.

BACKGROUND OF THE INVENTION

The present invention relates to treatment of inflammatory conditionssuch as colitis and hepatitis.

Human inflammatory bowel disease (IBD), such as Crohn's disease (CD) orulcerative colitis (UC), are characterized by inflammation in the largeand/or small intestine associated with uncontrolled innate and adaptiveimmunity against normal constituents, including commensal bacteria andvarious microbial products (Fiocchi et al., Gastroenterology,115:182-205 (1998); McKay, D. M., Can. J. Gastroenterology, 13:509-516(1999); Sartor, Res Immunol., 148:567-576 (1997); Cong et al., J. Exp.Med., 187:855-864 (1998); Maaser, et al., Z Gastroenterol., 40:525-529(2002)). Imbalance of pro-inflammatory cytokines in innate immunity hasbeen demonstrated to play a role in the pathogenesis of IBD (Reineckeret al., Clin. Exp. Immunol., 94:174-181 (1993)). Disregulated CD4 Tcells in adaptive immunity have also been postulated to play a role inthe pathogenesis of IBD (Fuss et al., J. Immunol., 157:1261-1270 (1996);Braegger et al., Ann. Allergy, 72:135-141 (1994); Monteleone et al.,Gastroenterology, 112:1169-78 (1997); Parronchi et al., Am. J. Pathol.150:823-832 (1997)). Responding T cells exhibit a T helper type 1 (Th1)phenotype capable of producing interferon (IFN)-gamma in CD whereas Th2cytokines are closely associated with UC (Fuss et al., J. Immunol.,157:1261-1270 (1996); Braegger et al., Ann. Allergy, 72:135-141 (1994);Monteleone et al., Gastroenterology, 112:1169-78 (1997); Parronchi etal., Am. J. Pathol. 150:823-832 (1997)).

A widely used animal model for UC is dextran sodium sulfate(DSS)-induced colitis. Mice that are exposed to DSS in their drinkingwater develop an inflammation of the colon displaying symptoms such asdiarrhea, rectal bleeding, and weight loss. The histological phenotypeof the acute phase of DSS-induced colitis is characterized by epithelialcell lesions and acute inflammation mainly consisting of infiltratinggranulocytes and macrophages (Cooper et al., Lab Invest, 69:238-249(1993); Okayasu et al., Gastroenterology, 98:694-702 (1990)).Lymphocytes have been described as not necessary for the acuteinflammatory phase of DSS-induced colitis. Mice lacking T cells,however, do not fully recover from colitis (Dieleman et al.,Gastroenterology, 107:1643-1652 (1994); Tsuchiya et al., J. Immunol.,171:5507-5513 (2003)). Exposure of animals to several cycles of DSSresults in the development of chronic colitis that is associated withlymphocyte infiltrates of CD4 T lymphocytes and B cells (Dieleman etal., Clin. Exp. Immunol., 114:385-391 (1998); Teramoto et al., Clin.Exp. Immunol., 139:421-428 (2005)). A role for T lymphocytes in theremission of DSS-induced colitis is not known.

In addition to cytokine expression, chemokines and their receptors alsocontribute to the regulation of intestinal immune responses and mucosalinflammation. The CC chemokine receptors CCR2 and CCR5 are involved inboth monocyte-mediated and macrophage-mediated immune responses, and inthe regulation of T cell migration and activation. Mice deficient inCCR2 or CCR5 are protected from DSS-induced colitis (Andres et al., J.Immunol., 164:6303-6312 (2000)). Certain cytokines (such as IL-1β)contribute to disease progression and tissue damage in humaninflammatory bowel disease.

T cell-mediated immune responses play a role in the development andprogression of various liver diseases, including autoimmune hepatitis,viral infection, and alcoholic hepatitis (Heneghan et al., Hepatology,35:7-13 (2002); Bogdanos et al., Dig. Liver Dis., 32:440-446 (2000);Chang, K. M. et al., Hepatology, 33:267-276 (2001); Chedid et al.,Gastroenterology, 105:254-266 (1993); Kita et al., Gastroenterology,120:1485-1501 (2001); Rehermann et al., Curr. Top. Microbiol. Immunol.,242:299-325 (2000); Eggink et al., Clin. Exp. Immunol., 50:17-24(1982)). Concanavalin A (ConA)-induced hepatitis is a murine model ofautoimmune or viral hepatitis that shares several pathologicalproperties with human autoimmune hepatitis and viral hepatitis (Lohr etal., Hepatology, 24:1416-1421 (1996)). ConA-induced hepatitis also hasbeen used as a model of T cell-mediated liver injury (Tiegs et al., A.J. Clin. Invest., 90:196-203 (1992)). These liver diseases areassociated with the infiltration of various lymphocyte subsets includingactivated T cells. The infiltration of CD4 T cells into the liver isinvolved in human autoimmune and virus hepatitis (Chang et al.,Hepatology, 33:267-276, 23 (2001); Rehermann et al., Curr. Top.Microbiol. Immunol., 242:299-325 (2000); Eggink et al., Clin. Exp.Immunol., 50:17-24 (1982)). T cells have a documented role in thepathogenesis of ConA-induced hepatitis including the induction andeffector phases (Tiegs et al., J. Clin. Invest., 90:196-203 (1992);Kusters et al., Gastroenterology, 111:462-471 (1996); Mizuhara et al.,Hepatology, 23:1608-1615 (1996); Toyonaga et al., Proc. Natl. Acad. Sci.U.S.A., 91: 614-618 (1994)). Pre-treatment with T cell-specificantibodies or immunosuppressive agents, such as anti-Thy-1, anti-CD4mAb, FK506, or cyclosporine A, inhibited ConA-induced hepatitis,indicating that CD4 T cells and their activation of TCR-mediatedsignaling are involved in the induction of ConA-induced hepatitis (Tiegset al., J. Clin. Invest., 90:196-203 (1992)). In addition, IFN-gammaappears to be involved in the development of ConA-induced hepatitis(Kusters et al., Gastroenterology, 111:462-471 (1996); Mizuhara et al.,Hepatology, 23:1608-1615 (1996); Toyonaga et al., Proc. Natl. Acad. Sci.U.S.A., 91:614-618 (1994)). NKT cells and their production of IFN-gammaplay a role in the development of ConA-induced hepatitis (Kaneko et al.,J. Exp. Med., 191:105-114 (2000)).

CD69 (also known as early T cell activation antigen p60; see MendelianInheritance in Man I.D. No. *107273) is a type II membrane proteinexpressed as a homodimer of heavily glycosylated subunits (Ziegler etal., Eur. J. Immunol., 23:1643-1648 (1993)). Both T and B cells expressCD69 within a few hours after stimulation. CD69 is an early activationmarker antigen of lymphocytes (Testi et al., Immunol. Today, 15:479-483(1994)). Freshly prepared thymocytes undergoing selection events expressCD69 (Nakayama et al., J. Immunol., 168:87-94 (2002); Feng et al., Int.Immunol., 14:535-544 (2002)). There may be regulatory roles for CD69expression in T cell development in the thymus, as well as a mild effecton B cell development (Lauzurica et al., Blood, 95:2312-2320 (2000)).Constitutive expression of CD69 has been noted in platelets, andactivated neutrophils and eosinophils express CD69 on their cellsurface. CD69 may have regulatory roles in a collagen-induced arthritismodel and an anti-collagen antibody-induced arthritis model, possiblyinvolving multiple target processes (Sancho et al., J. Clin. Invest.,112:872-882 (2003); Murata et al., Int. Immunol., 15:987-992 (2003)).The role of CD69 in other inflammatory models, such as in allergicairway inflammation, however, has not been ascertained. A function ofCD69 in lymphocyte trafficking has been proposed (Shiow et al., Nature,440:540-544 (2006)). A role for CD69 in the development of otherinflammatory conditions, such as colitis and hepatitis, is unknown.

Accordingly, a need exists for new therapies for treating inflammatoryconditions, such as colitis and hepatitis. New anti-inflammatorytherapies employing one or more therapeutic compounds against a specificmolecular target are sought.

All the patents and publications mentioned above and throughout areincorporated in their entirety by reference herein.

SUMMARY OF THE PRESENT INVENTION

It is therefore a feature of the present invention to provide methodsfor treating or reducing the susceptibility to inflammatory conditionsgenerally.

Another feature of the present invention is to provide methods fortreating or reducing the susceptibility to inflammatory conditionsthrough the inhibition of CD69.

A further feature of the present invention is to provide methods fortreating or reducing the susceptibility to colitis and hepatitisspecifically.

Another feature of the present invention is the inhibition of CD69 incombination with one or more additional therapies to treat or reduce thesusceptibility to an inflammatory condition, such as colitis orhepatitis.

Additional features and advantages of the present invention will be setforth in part in the description that follows, and in part will beapparent from the description, or may be learned by practice of thepresent invention. The objectives and other advantages of the presentinvention will be realized and attained by means of the elements andcombinations particularly pointed out in the description and appendedclaims.

To achieve these and other advantages, and in accordance with thepurposes of the present invention, as embodied and broadly describedherein, the present invention relates to a method of treating orreducing the susceptibility to at least one inflammatory conditioninvolving administering at least one CD69 antagonist to a subject,wherein the subject has been diagnosed with at least one inflammatorycondition, or susceptibility to the same, including colitis, hepatitis,a susceptibility to colitis, a susceptibility to hepatitis, or anycombination thereof, in an amount effective to treat or reduce thesusceptibility to the at least one inflammatory condition.

Any number of CD69 antagonists may be employed, alone, or in combinationwith other therapies for treating or reducing the susceptibility to atleast one inflammatory condition. CD69 antagonists can include one ormore of an anti-CD69 antibody, an anti-CD69 aptamer, a CD69 mRNAantagonist, a small molecule pharmaceutical, or any combination thereof.Administration of at least one CD69 antagonist, alone, or in combinationwith one or more therapies, can be continued when one or more symptomsof an inflammatory condition, such as colitis or hepatitis persists, orcan be discontinued when such symptoms decrease or disappear. Acombination or two or more CD69 antagonists or one or more CD69antagonists with one or more therapies can synergistically decrease oneor more symptoms of an inflammatory condition.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are intended to provide a further explanation of the presentinvention, as claimed.

The accompanying drawings, which are incorporated in and constitute apart of this application, illustrate some of the embodiments of thepresent invention and together with the description, serve to explainthe principles of the present invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A is a graph that shows data for inhibition of DSS-induced colitisin CD69 knock-out (KO) mice. Colitis is induced by giving animals 4% DSSin drinking water for 7 days followed by normal drinking water. Survivalrates of CD69−/−, CD69+/− and wild-type (WT) mice during DSS-inducedacute colitis are provided. Survival is recorded daily (n=10 per group).

FIG. 1B is a graph that shows further data for the experiment describedfor FIG. 1A. Changes in the disease activity index over the course ofDSS treatment in CD69−/−, CD69+/− and WT mice are shown.

FIG. 1C is a graph that shows further data for the experiment describedfor FIG. 1A. Changes in the body weight (%) over the course of DSStreatment in CD69−/−, CD69+/− and WT mice are shown. Data are presentedas the mean with SEM (n=10 per group; *p<0.05).

FIG. 2A is a graph that shows data demonstrating that DSS-inducedcolocaecal damage is reduced in CD69 KO mice. At day 8, colon length ofDSS-exposed WT and CD69 KO mice and controls is measured. Data arepresented as the mean with SEM (n=12 per group; *p<0.01).

FIG. 2B are photographs of colons and shows further data for theexperiment described for FIG. 2A. Representative gross appearance of thecolon from each group is shown.

FIG. 2C shows further data for the experiment described for FIG. 2A.Histological sections of inflamed colons are shown. Colons are taken atday 8 from WT and CD69 KO mice receiving DSS in drinking water. Sectionsare prepared and stained with H&E. Original magnification ×200.

FIG. 2D shows further data for the experiment described for FIG. 2A.Immunohistochemical staining of CD69-positive cells in the colonictissues of DSS-treated WT mice is shown (original magnification ×400).

FIG. 3A is a graph that shows data demonstrating that DSS-inducedcolitis was restored by cell transfer of wild-type CD4 T cells intoCD69-KO mice. Survival rates of WT and CD69 KO mice without celltransfer, and CD69-KO mice transferred with whole spleen cells, CD4 Tcells or neutrophils during DSS-induced acute colitis are shown.Survival is recorded daily (n=5 per group).

FIG. 3B is a graph that shows further data for the experiment describedfor FIG. 3A. Changes in the disease activity index over the course ofDSS treatment in WT and CD69 KO mice without cell transfer, and CD69-KOmice transferred with whole spleen cells, CD4 T cells or neutrophils areshown. Data are presented as the mean with SD (n=5 per group; *p<0.05).

FIG. 3C is a graph that shows further data for the experiment describedfor FIG. 3A. Changes in the body weight (%) over the course of DSStreatment in WT and CD69 KO mice without cell transfer, and CD69-KO micetransferred with whole spleen cells, CD4 T cells or neutrophils areshown. Data are presented as the mean with SD (n=5 per group; *p<0.05).

FIG. 3D is a graph that shows further data for the experiment describedfor FIG. 3A. Colon length of controls and DSS-exposed WT and CD69 KOmice without cell transfer, and CD69-KO mice transferred with wholespleen cells, CD4 T cells or neutrophils at day 8 is measured. Data arepresented as the mean with SE (n=5 per group; *p<0.01).

FIG. 3E shows further data for the experiment described for FIG. 3A.Histological sections of inflamed colons are prepared. Colons are takenat day 8 from controls and DSS-exposed CD69 KO mice without celltransfer, and CD69-KO mice transferred with whole spleen cells, CD4 Tcells or neutrophils. Sections are fixed and stained with H&E (Originalmagnification ×200).

FIG. 4A is a graph that shows data demonstrating the effect of in vivotreatment with anti-CD69 mAb on DSS-induced colitis. WT BALB/c mice aretreated with anti-CD69 mAb or control hamster IgG on day 0, Survival ofeach group during DSS-induced colitis is recorded daily (n=5 per group).

FIG. 4B is a graph that shows further data for the experiment describedfor FIG. 4A. Changes in the disease activity index over the course ofDSS treatment in WT mice treated with anti-CD69 mAb or control hamsterIgG are shown. Data are presented as the mean with SD (n=5 per group;*p<0.05).

FIG. 4C is a graph that shows further data for the experiment describedfor FIG. 4A. Changes in the body weight (%) (over the course of DSStreatment in WT mice treated with anti-CD69 mAb or control hamster IgGare shown. Data are presented as the mean with SD (n=5 per group;*p<0.05).

FIG. 4D is a bar graph that shows further data for the experimentdescribed for FIG. 4A. Colon length of controls and DSS-exposed WT micetreated with anti-CD69 mAb or control hamster IgG are shown. Data arepresented as the mean with SE (n=5 per group; *p<0.01).

FIG. 4E shows further data for the experiment described for FIG. 4A.Histological sections are prepared. Colons are taken at day 8 fromcontrols and DSS-exposed WT mice treated with anti-CD69 mAb or controlhamster IgG. Sections are fixed and stained with H&E (originalmagnification ×200).

FIG. 5 is a series of bar graphs that show data demonstrating theexpression of cytokines, chemokines and their receptor mRNA in thecolons of DSS-treated mice. Five mice per group are treated as describedin experiment described for FIG. 1A. Mice are sacrificed at day 8 andthe colon tissues are harvested. Whole colonic RNA is isolated, reversetranscribed into cDNA and the expressions of IL-1β, IL-6, IL-10, CCR2,CCR3, CCL2, CCL4 and CCL5 are determined by real-time quantitative PCR.The amounts of mRNAs are normalized by the HPRT signal in the respectivesample. PCR is performed in triplicate for each sample. There are nosignificant increases in mRNA expression in either the untreated WT orCD69 KO mice. The data presented are representative of three individualexperiments.

FIG. 6A is a set of bar graphs that show data demonstrating theattenuation of ConA-induced hepatitis in CD69-deficient mice. Plasma ASTand ALT levels in CD69KO and WT mice 12 h after ConA (10 mg/kg, i.v.)injection are shown. The results are expressed as mean±SD (n=5;*p<0.05), compared with WT mice.

FIG. 6B shows further data for the experiment described for FIG. 6A. Thelivers are collected 12 hours after ConA injection, and the liver damageis evaluated by H&E staining.

FIG. 7A is a set of bar graphs that show data demonstrating theattenuation of ConA-induced hepatitis by anti-CD69 antibodyadministration. Plasma AST and ALT levels in ConA-induced hepatitis inmice are shown. WT C57BL/6 mice are treated with anti-CD69 mAb (400μg/mouse) or control hamster IgG intraperitoneally 30 min before theinjection of ConA. The plasma is collected 12 h after ConA injection.The results are expressed as mean±SD (n=5; *p<0.05).

FIG. 7B shows further data for the experiment described for FIG. 7A. Thelivers are collected 12 h after ConA injection and the liver damage isevaluated by H&E staining.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

In accordance with the present invention, a method of treating orreducing the susceptibility to at least one inflammatory condition isprovided involving administering at least one CD69 antagonist to asubject, wherein the subject has been diagnosed with at least oneinflammatory condition, or a susceptibility to the same, includingcolitis, hepatitis, a susceptibility to colitis, a susceptibility tohepatitis, or any combination thereof, in an amount effective to treator reduce the inflammation and/or the susceptibility to the at least oneinflammatory condition.

When the condition is colitis, the method can further include monitoringat least one symptom of colitis. Examples of colitis symptoms includeepithelial cell lesions, acute inflammation, diarrhea, rectal bleeding,and/or weight loss. Colitis symptoms can include abdominal pain,abdominal cramps, an urgent need to have a bowel movement, fever, and/orloss of hunger. The method can further include continuing administrationif the at least one symptom persists, falls within a given range, orremains above or below a threshold value; or discontinuingadministration if the at least one symptom fails to persist, fails tofall within a given range, or no longer remains above or below athreshold value. Any suitable test can be used for diagnosis of ormonitoring of colitis or a susceptibility to the same. Examples of suchtests include colonoscopy, biopsy, barium enema X-ray, abdominal X-ray,blood tests for infection or inflammation, stool sample analysis forblood, infection, and/or white blood cell levels and antigens.

Administration of the CD69 antagonist can be combined with one or moreanti-colitis therapies. Examples of additional anti-colitis therapiescan include medications such as at least one anti-diarrheal medication,at least one aminosalicylate, at least one corticosteroid, at least oneimmunosuppressant, or any combination thereof. Examples ofanti-diarrheal medications include loperamide or salts thereof such asloperamide hydrochloride. Aminosalicylates can include salts thereofsuch as aminosalicylate sodium. Corticosteroids can include prednisone,prednisolone, methylprednisolone, dexamethasone, salts thereof, orcombinations thereof. Immunosuppressants can include, for example,cyclosporins, azathioprines, interleukin-2 inhibiting monoclonalantibodies (basiliximab, daclizumab, and muromonab), salts thereof, orcombinations thereof.

Any type or types of colitis can be the subject of treatment orreduction in susceptibility. For example, the colitis can be anulcerative colitis. As colitis is a form of inflammatory bowel disease(IBD), treatment of IBD or a reduction in susceptibility to IBD byadministration of a CD69 antagonist is an aspect of the presentinvention. Crohn's disease is a form of inflammatory bowel disease. Afeature of the present invention is the treatment of and/or reduction ofsusceptibility to Crohn's disease as described herein in respect tocolitis. Whereas colitis is usually associated with the colon andrectum, Crohn's disease can affect the digestive tract more generally.

When the condition is hepatitis, the method can further includemonitoring at least one symptom of hepatitis. Examples of hepatitissymptoms include at least one of acute inflammation, raised level ofaspartate aminotransferase (AST), and/or raised level of alanineaminotransferase (ALT). The method can further include continuingadministration if the at least one symptom persists, falls within agiven range, or remains above or below a threshold value; ordiscontinuing administration if the at least one symptom fails topersist, fails to fall within a given range, or no longer remains aboveor below a threshold value.

Any type or types of hepatitis can be the subject of treatment orreduction in susceptibility. For example, the hepatitis can be hepatitisA, hepatitis B, hepatitis C, or any combination thereof. Symptoms ofhepatitis A can include fatigue, fever, muscle soreness, headache,abdominal pain, nausea, appetite loss, weight loss, jaundice, or anycombination thereof. Symptoms of hepatitis B can include jaundice,fatigue, mild fever, headache, appetite loss, nausea, vomiting,abdominal pain, diarrhea, constipation, muscle aches, joint pain, skinrash, or any combination thereof. Symptoms of hepatitis C can includefatigue, joint pain, abdominal pain, itchiness, muscle soreness,jaundice, or any combination thereof.

Diagnosis for, or monitoring of, hepatitis or susceptibility to the samecan include one or more blood test to study liver function (heightenedbilirubin levels, decreased albumin levels, abnormal prothrombin(clotting) time, anti-hepatitis A, B, or C antibodies or antigens, andthe like). Diagnosis for, and monitoring of, hepatitis or susceptibilityto the same can include one or more tests for heightened enzyme levelsin the blood, such as alanine aminotransferase (ALT), aspartateaminotransferase (AST), alkaline phosphatase (ALP), lactic dehydrogenase(LDH). Liver biopsies can also be employed. Pathogenic nucleic acidssuch as hepatitis viral RNA can be tested for diagnosis purposes andmonitoring.

Administration of the CD69 antagonist can be combined with one or moreanti-hepatitis therapies. Examples of additional anti-hepatitis Atherapies can include at least one vaccine, at least one immunoglobulin,or any combination thereof. Examples of additional anti-hepatitis Btherapies can include at least one vaccine, at least one interferon, atleast one nucleoside reverse transcriptase inhibitor, or any combinationthereof. Interferons can include interferon alfa-2a, interferon alfa-2b,any pegylation thereof, or any combination thereof. Nucleoside reversetranscriptase inhibitors (NRTIs) can include adefovir, entecavir,lamivudine, telbivudine, any salt thereof, any prodrug thereof, or anycombination thereof. Examples of additional anti-hepatitis C therapiescan include at least one vaccine, at least one peginterferon, ribavirin,and/or any combination thereof.

The CD69 antagonist administered can include an antibody or apolypeptide containing an antigen-binding fragment of the antibody. Theantibody administered can include a monoclonal or polyclonal antibody.The antibody or the polypeptide administered can include anantigen-binding fragment of the antibody that binds to an extracellulardomain of CD69. The antibody or polypeptide can inhibit the activity ofCD69. The CD69 antagonist can contain a small molecule (low molecularweight) drug, salts, prodrugs, or combinations thereof. The antagonistcan inhibit CD69 activity. Any CD69 amino acid sequence can be employedfor producing antibodies or aptamers that bind CD69. Examples of suchsequences include those sequences, versions thereof, portions thereof,or combinations thereof with Accession Nos. BAF84558, ABM87473,ABM84101, EAW96123, EAW96122, Q53ZX0, AAO63584, AAH07037, NP_(—)001772,Q07108, CAA83017, CAA80298, or AAB46359.

Antibodies specific for an epitope of CD69, for example an extracellulardomain of CD69, and polypeptides containing antigen binding fragmentsthereof are provided as well as methods, uses, compositions, and kitsemploying the same. A method of forming an antibody specific to anepitope of CD69 or a polypeptide or a fragment thereof is provided. Sucha method can contain providing a nucleic acid encoding a CD69polypeptide or a polypeptide containing an immunologically specificepitope thereof; expressing a CD69 polypeptide comprising a CD69 aminoacid sequence or a polypeptide containing an immunologically specificepitope thereof from the isolated nucleic acid; and generating anantibody specific to the polypeptide obtained or a polypeptidecontaining an antigen binding fragment thereof. An antibody orpolypeptide comprising an antigen binding fragment thereof produced bythe aforementioned method is provided. An isolated antibody or isolatedpolypeptide containing an antigen binding fragment thereof thatspecifically binds an epitope of CD69 containing a CD69 amino acidsequence is provided. Such an antibody can be generated using anyacceptable method(s) known in the art. The antibodies as well as kits,methods, and/or other aspects of the present invention employingantibodies can include one or more of the following: a polyclonalantibody, a monoclonal antibody, a chimeric antibody, a single-chainantibody, a monovalent antibody, a diabody, and/or a humanized antibody.

Naturally occurring antibody structural units typically contain atetramer. Each such tetramer can be composed of two identical pairs ofpolypeptide chains, each pair having one full-length light” (forexample, about 25 kDa) and one full-length “heavy” chain (for example,about 50-70 kDa). The amino-terminal portion of each chain typicallyincludes a variable region of about 100 to 110 or more amino acids thattypically is responsible for antigen recognition. The carboxy-terminalportion of each chain typically defines a constant region that may beresponsible for effector function. Human light chains are typicallyclassified as kappa and lambda light chains. Heavy chains are typicallyclassified as mu, delta, gamma, alpha, or epsilon, and define theantibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG hasseveral subclasses, including, but not limited to, IgG1, IgG2, IgG3, andIgG4. IgM has subclasses including, but not limited to, IgM1 and IgM2.IgA is similarly subdivided into subclasses including, but not limitedto, IgA1 and IgA2. In light and heavy chains, the variable and constantregions can be joined by a “J” region of about 12 or more amino acids,with the heavy chain also including a “D” region of about 10 or moreamino acids. See, e.g., Fundamental Immunology Ch. 7 (Paul, W., ed., 2nded. Raven Press, N.Y. (1989)) (incorporated by reference in its entiretyfor all purposes). The variable regions of each light/heavy chain pairtypically form the antigen binding site.

The variable regions typically exhibit the same general structure ofrelatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions orCDRs. The CDRs from the two chains of each pair typically are aligned bythe framework regions, which may enable binding to a specific epitope.From N-terminal to C-terminal, both light and heavy chain variableregions typically contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3and FR4. The assignment of amino acids to each domain is typically inaccordance with the definitions of Kabat Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.(1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987);Chothia et al., Nature 342:878-883 (1989).

“Antibody fragments” include a portion of an intact antibody, such asthe antigen binding or variable region of the intact antibody. Examplesof antibody fragments include Fab, Fab1, F(ab′)2, and Fv fragments;diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10):1057-1062 [1995]); single-chain antibody molecules; and multispecificantibodies formed from antibody fragments. Papain digestion ofantibodies produces two identical antigen-binding fragments, called“Fab” fragments, each with a single antigen-binding site, and a residual“Fc” fragment, a designation reflecting the ability to crystallizereadily. Pepsin treatment yields an F(ab′)2 fragment that has twoantigen-combining sites and is still capable of cross-linking antigen.“Fv” is an antibody fragment which contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light-chain variable domain in tight, non-covalentassociation. A single variable domain (or half of an Fv containing onlythree CDRs specific for an antigen) can recognize and bind an antigen.“Single-chain Fv” or “sFv” antibody fragments include the VH and VLdomains of the antibody, wherein these domains are present in a singlepolypeptide chain. The Fv polypeptide can further contain a polypeptidelinker between the VH and VL domains which enables the sFv to form thedesired structure for antigen binding. For a review of sFv, seePluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

Antibodies can be used as probes, therapeutic treatments and other uses.Antibodies can be made by injecting mice, rabbits, goats, or otheranimals with the translated product or synthetic peptide fragmentsthereof. These antibodies are useful in diagnostic assays or as anactive ingredient in a pharmaceutical composition.

The antibody or polypeptide administered can be conjugated to afunctional agent to form an immunoconguate. The functional agent can bea cytotoxic agent such as a chemotherapeutic agent, a toxin (e.g., anenzymatically active toxin of bacterial, fungal, plant, or animalorigin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate), an antibiotic, a nucleolytic enzyme, or any combinationthereof. Chemotherapeutic agents can be used in the generation ofimmunoconjugates, e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents, enzymes,and/or fragments thereof, such as nucleolytic enzymes, antibiotics, andtoxins such as small molecule toxins or enzymatically active toxins ofbacterial, fungal, plant or animal origin, including fragments and/orvariants thereof, and the various antitumor or anticancer agentsdisclosed below. Enzymatically active toxins and fragments thereof thatcan be used include, for example, diphtheria A chain, nonbinding activefragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricotheeenes.Any appropriate radionucleotide or radioactive agent known in the art orare otherwise available can be used to produce radioconjugatedantibodies.

Conjugates of the antibody and cytotoxic agent can be made using avariety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP); iminothiolane(IT); bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL); active esters (such as disuccinimidyl suberate);aldehydes (such as glutareldehyde); bis-azido compounds (such asbis(p-azidobenzoyl) hexanediamine); bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine); diisocyanates (such astolyene 2,6-diisocyanate); bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene); maleimidocaproyl (MC);valine-citrulline, dipeptide site in protease cleavable linker (VC);2-amino-5-ureido pentanoic acid PAB=p-aminobenzylcarbamoyl (“selfimmolative” portion of linker) (Citrulene); N-methyl-valine citrullinewhere the linker peptide bond has been modified to prevent its cleavageby cathepsin B (Me); maleimidocaproyl-polyethylene glycol, attached toantibody cysteines; N-Succinimidyl 4-(2-pyridylthio)pentanoate (SPP);and N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate(SMCC). For example, a ricin immunotoxin can be prepared as described inVitetta et al., Science, 238: 1098 (1987). Carbon-14-labeled1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacctic acid(MX-DTPA) is an exemplary chelating agent for conjugation ofradionucleotide to the antibody, see WO 94/11026. The antibody can beconjugated to a “receptor” (such as streptavidin) for utilization intumor pre-targeting wherein the antibody-receptor conjugate isadministered to the subject, followed by removal of unbound conjugatefrom the circulation using a clearing agent and then administration of a“ligand” (e.g., avidin) that is conjugated to a cytotoxic agent (e.g., aradionucleotide).

The antibodies of the present invention can be coupled directly orindirectly to a detectable marker by techniques well known in the art. Adetectable marker is an agent detectable, for example, by spectroscopic,photochemical, biochemical, immunochemical, or chemical means. Usefuldetectable markers include, but are not limited to, fluorescent dyes,chemiluminescent compounds, radioisotopes, electron-dense reagents,enzymes, colored particles, biotin, or dioxigenin. A detectable markeroften generates a measurable signal, such as radioactivity, fluorescentlight, color, or enzyme activity. Antibodies conjugated to detectableagents may be used for diagnostic or therapeutic purposes. Examples ofdetectable agents include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. Thedetectable substance can be coupled or conjugated either directly to theantibody or indirectly, through an intermediate such as, for example, alinker known in the art, using techniques known in the art. See, e.g.,U.S. Pat. No. 4,741,900, describing the conjugation of metal ions toantibodies for diagnostic use. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, beta-galactosidase, andacetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride and phycoerythrin; an example of a luminescent materialincludes luminol; examples of bioluminescent materials includeluciferin, and aequorin.

Antibodies useful in practicing the present invention can be prepared inlaboratory animals or by recombinant DNA techniques using the followingmethods. Polyclonal antibodies can be raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the gene productmolecule or fragment thereof in combination with an adjuvant such asFreund's adjuvant (complete or incomplete). To enhance immunogenicity,it may be useful to first conjugate the gene product molecule or afragment containing the target amino acid sequence to a protein that isimmunogenic in the species to be immunized, e.g., keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsininhibitor using a bifunctional or derivatizing agent, for example,maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteineresidues), N-hydroxysuccinimide (through lysine residues),glutaraldehyde, succinic anhydride, SOCl, etc. Alternatively,immunogenic conjugates can be produced recombinantly as fusion proteins.

Animals can be immunized against the immunogenic conjugates orderivatives (such as a fragment containing the target amino acidsequence) by combining about 1 mg or about 1 microgram of conjugate (forrabbits or mice, respectively) with about 3 volumes of Freund's completeadjuvant and injecting the solution intradermally at multiple sites.Approximately 7 to 14 days later, animals are bled and the serum isassayed for antibody titer. Animals are boosted with antigen repeatedlyuntil the titer plateaus. The animal can be boosted with the samemolecule or fragment thereof as was used for the initial immunization,but conjugated to a different protein and/or through a differentcross-linking agent. In addition, aggregating agents such as alum can beused in the injections to enhance the immune response.

The antibody administered can include a chimeric antibody. The antibodyadministered can include a humanized antibody. The antibody administeredcan include a completely humanized antibody. The antibodies can behumanized or partially humanized. Non-human antibodies can be humanizedusing any applicable method known in the art. A humanized antibody canbe produced using a transgenic animal whose immune system has beenpartly or fully humanized. Any antibody or fragment thereof of thepresent invention can be partially or fully humanized. Chimericantibodies can be produced using any known technique in the art. See,e.g., U.S. Pat. Nos. 5,169,939; 5,750,078; 6,020,153; 6,420,113;6,423,511; 6,632,927; and 6,800,738.

The antibody administered can include a monoclonal antibody, that is,the anti-CD69 antibodies of the present invention that can be monoclonalantibodies. Monoclonal antibodies can be prepared using hybridomamethods, such as those described by Kohler and Milstein, Nature, 256:495(1975). In a hybridoma method, a mouse, hamster, or other appropriatehost animal, is typically immunized with an immunizing agent to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the immunizing agent. Alternatively, thelymphocytes can be immunized in vitro. Monoclonal antibodies can bescreened as are described, for example, in Harlow & Lane, Antibodies, ALaboratory Manual, Cold Spring Harbor Press, New York (1988); Goding,Monoclonal Antibodies, Principles and Practice (2d ed.) Academic Press,New York (1986). Monoclonal antibodies can be tested for specificimmunoreactivity with a translated product and lack of immunoreactivityto the corresponding prototypical gene product.

Monoclonal antibodies can be prepared by recovering spleen cells fromimmunized animals and immortalizing the cells in conventional fashion,e.g., by fusion with myeloma cells. The clones are then screened forthose expressing the desired antibody. The monoclonal antibodypreferably does not cross-react with other gene products. After thedesired hybridoma cells are identified, the clones can be subcloned bylimiting dilution procedures and grown by standard methods. Suitableculture media for this purpose include, for example, Dulbecco's ModifiedEagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cellscan be grown in vivo as ascites in a mammal. The monoclonal antibodiessecreted by the subclones can be isolated or purified from the culturemedium or ascites fluid by conventional immunoglobulin purificationprocedures such as, for example, protein A-Sepharose, hydroxylapatitechromatography, gel electrophoresis, dialysis, or affinitychromatography.

The monoclonal antibodies can also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the present invention can be readily isolatedand sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies). The hybridomacells of the present invention can serve as a preferred source of suchDNA. Once isolated, the DNA may be placed into expression vectors, whichare then transfected into host cells such as simian COS cells, Chinesehamster ovary (CHO) cells, or myeloma cells that do not otherwiseproduce immunoglobulin protein, to obtain the synthesis of monoclonalantibodies in the recombinant host cells. The DNA also can be modified,for example, by substituting the coding sequence for human heavy andlight chain constant domains in place of the homologous murine sequencesor by covalently joining to the immunoglobulin coding sequence all orpart of the coding sequence for a non-immunoglobulin polypeptide. Such anon-immunoglobulin polypeptide can be substituted for the constantdomains of an antibody of the present invention, or can be substitutedfor the variable domains of one antigen-combining site of an antibody ofthe invention to create a chimeric bivalent antibody. Preparation ofAntibodies Using Recombinant DNA Methods Such as the Phagemid DisplayMethod, can be accomplished using commercially available kits, as forexample, the Recombinant Phagemid Antibody System available fromPharmacia (Uppsala, Sweden), or the SurfZAP™ phage display system(Stratagene Inc., La Jolla, Calif.).

Also included in the present invention are hybridoma cell lines,transformed B cell lines, and host cells that produce the monoclonalantibodies of the present invention; the progeny or derivatives of thesehybridomas, transformed B cell lines, and host cells; and equivalent orsimilar hybridomas, transformed B cell lines, and host cells. Examplesof hybridoma cells lines include without limitation H1.2F3 (BioLegend,Inc., San Diego, Calif.).

The antibodies can be diabodies. The term “diabodies” refers to smallantibody fragments with two antigen-binding sites, which fragmentsinclude a heavy-chain variable domain (VH) connected to a light-chainvariable domain (VL) in the same polypeptide chain (Vn-VL). By using alinker that is too short to allow pairing between the two domains on thesame chain, the domains can be forced to pair with the complementarydomains of another chain and create two antigen-binding sites. Diabodiesare described more fully in, for example, EP 404,097; WO 93/11161; andHollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

The antibody administered can include a single-chain antibody. Theantibodies can be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain can be truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart.

The antibodies can be bispecific. Bispecific antibodies thatspecifically bind to one protein and that specifically bind to otherantigens relevant to pathology and/or treatment are produced, isolated,and tested using standard procedures that have been described in theliterature. [See, e.g., Pluckthun & Pack, Immunotechnology, 3:83-105(1997); Carter, et al., J. Hematotherapy, 4:463-470 (1995); Renner &Pfreundschuh, Immunological Reviews, 1995, No. 145, pp. 179-209;Pfreundschuh U.S. Pat. No. 5,643,759; Segal, et al., J. Hematotherapy,4:377-382 (1995); Segal, et al., Immunobiology, 185:390-402 (1992); andBolhuis, et al., Cancer Immunol. Immunother., 34:1-8 (1991)].

The antibodies disclosed herein can be formulated as immunoliposomes.Liposomes containing the antibody are prepared by methods known in theart. such as described in Epstein et al., Proc. Natl. Acad. Sci. USA,82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77: 4030(1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes withenhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.Particularly useful liposomes can be generated by the reverse-phaseevaporation method with a lipid composition containingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes can be extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem., 257:286-288 (1982) via a disulfide-interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome. See Gabizon et al, J. National Cancer Inst.,81(19): 1484 (1989).

The CD69 antagonist can include an aptamer that binds CD69. The aptamercan contain one or more of a nucleic acid, a RNA, a DNA, and an aminoacid. Aptamers can be selected and produced using any suitable techniqueor protocol. For example, oligonucleotide libraries with variableregions ranging from 18 to 50 nucleotides in length can be used astemplates for run-off transcription reactions to generate random poolsof RNA aptamers. This aptamer pool can then be exposed to unconjugatedmatrix to remove non-specific interacting species. The remaining pool isthen incubated with an immobilized target. The majority of aptamerspecies in this pool can have low affinity, for the target can be washedaway leaving a smaller, more specific pool bound to the matrix. Thispool can then be eluted, precipitated, reverse transcribed, and used asa template for run-off transcription. After five rounds of selection,aliquots can be removed that are cloned and sequenced. Selection can becontinued until similar sequences are reproducibly recovered.

Aptamer production can be performed using a bead-based selection system.In this process, a library of beads is generated in which each bead iscoated with a population of aptamers with identical sequences composedof natural and modified nucleotides. This bead library, which cancontain greater than 100,000,000 unique sequences, can be incubated witha peptide that corresponds to CD69, or a portion thereof, e.g., anextracellular domain, that is conjugated with a tag such as afluorescent dye. After washing, beads that demonstrate the highestbinding affinity can be isolated and aptamer sequences can be determinedfor subsequent synthesis.

The CD69 antagonist administered can include a mRNA antagonist. Examplesof mRNA antagonists can include at least one siRNA or at least oneribozyme. In accordance with the present invention, the CD69 antagonistcan be a therapeutic nucleic acid, such as a siRNA, can target a CD69nucleotide sequence, complements thereof, or any combination thereof.Any suitable CD69 sequence can be employed. CD69 target sequences of thesynthetic siRNAs can be designed against a human CD69 nucleotidesequence, or any portion or combination thereof, with Accession No.NM_(—)001781, NR_(—)026672, NR_(—)026671, AK303383, AK303174, AK291869,DQ896474, DQ893175, CH471094, AY238518, BC007037, AC007068, Z38109,Z30426, Z22576, or L07555, or recognize all or a subset of CD69transcript variants.

The CD69 antagonist employed in the present invention can cause adecrease in the expression of CD69. A CD69 mRNA antagonist is an exampleof such an antagonist. The CD69 antagonist can be a nucleic acid atleast 10 nucleotides in length that specifically binds to and iscomplementary to a target nucleic acid encoding CD69 or a complementthereof, wherein the administration of the CD69 antagonist involvesintroducing the nucleic acid into a cell of the subject. RNAinterference (RNAi) can be employed and the CD69 antagonist can be asmall interfering RNA (siRNA). The administration of the CD69 antagonistinvolves introducing into a cell of a subject, wherein the cell iscapable of expressing CD69 as an effective amount of a small interferingRNA (siRNA) nucleic acid for a time and under conditions sufficient tointerfere with expression of CD69. siRNA nucleic acids can includeoverhangs. That is, not all nucleotides need bind to the targetsequence. The siRNA nucleic acids can contain RNA. The siRNA nucleicacid can also contain DNA, that is, deoxyribonucleic acid nucleotides.Any type of suitable small interfering RNA can be employed. EndogenousmicroRNA (miRNA) can be employed. Other RNA interference agents that canbe used in accordance with the present invention include short hairpinRNA (shRNA), trans-acting siRNAs (tasiRNAs), repeat-associated siRNAs(rasiRNAs), small-scan (scn)RNAs, and Piwi-interacting (pi)RNAs. RNAinterference nucleic acids employed can be at least 10, at least 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,at least 35, and/or between 40-50 nucleotides in length. RNAi agent canalso include one or more deoxyribonucleotide. The RNAi agent, forexample, siRNA or shRNA, can be included as a cassette by a largernucleic acid construct such as an appropriate vector system. Examples ofsuch vectors systems include lentiviral and adenoviral vector systems.An example of a suitable system is described in Aagaard et al., Mol.Ther., 15(5):938-45 (2007). When present as part of a larger nucleicacid construct, the resulting nucleic acid can be longer than theincluded RNAi nucleic acid, for example, greater than 50 nucleotides inlength. The RNAi agent employed may or may not cleave the target mRNA.

In addition or in the alternative to RNA interference, other nucleicacid antagonists can be employed. The CD69 antagonist can be a ribozymethat specifically cleaves an RNA molecule transcribed from a geneencoding CD69, wherein the ribozyme contains a target substrate bindingsite, a catalytic sequence within the substrate binding site, whereinthe substrate binding site is complementary to a portion of an RNAmolecule transcribed from the CD69 gene. The CD69 antagonist can be anantisense nucleic acid containing a nucleotide sequence complementary toat least 8 nucleotides of a nucleic acid encoding CD69 or a complementthereof. The antisense nucleic acid can be complementary to a CD69sequence that is of sufficient length and sequence content such that theantisense nucleic acid does not crossreact with non-CD69 nucleotidesequences. Cross-reaction can occur but not cause a substantialdeleterious side effect.

The CD69 antagonist administered can include a small moleculepharmaceutical. For example, CD69-disabling small peptide mimetics canbe employed. Such mimetics can be constructed to resemble secondarystructural features of the targeted protein 69.

In accordance with the present invention, two or more CD69 antagonistscan be administered. At least one CD69 antagonist can be administered incombination with one or more additional therapies directed to colitis,hepatitis, a susceptibility to colitis, a susceptibility to hepatitis,or any combination thereof. The administration of two or more therapies,including one or more CD69 antagonists, can be simultaneous, sequential,or in combination. Accordingly, when two or more therapies areadministered, they need not be administered simultaneously or in thesame way or in the same dose. When administered simultaneously, the twoor more therapies can be administered in the same composition or indifferent compositions. The two or more therapies can be administeredusing the same route of administration or different routes ofadministration. When administered at different times, the therapies canbe administered before or after each other. Administration order of thetwo or more therapies can be alternated. The respective doses of the oneor more therapies can be varied over time. The type of one or moretherapy can be varied over time. When administered at separate times,the separation of the two or more administrations can be any timeperiod. If administered multiple times, the length of the time periodcan vary. The separation between administration of the two or more twoor more therapies can be 0 seconds, 1 second, 5 seconds, 10 seconds, 30seconds, 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30,minutes, 45 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 4hours, 5 hours, 7.5 hours, 10 hours, 12 hours, 15 hours, 18 hours, 21hours, 24 hours, 1.5 days, 2 days, 3 days, 4 days, 5 days, 6 days, 7days, 10 days, 2 weeks, 3 weeks, 4 weeks, one month, 6 weeks, 8 weeks,three months, six months, 1 year or longer.

Two or more CD69 antagonists can act synergistically to treat or reducethe susceptibility to the at least one inflammatory condition. At leastone CD69 antagonist and the one or more additional therapies can actsynergistically to treat or reduce the susceptibility to the at leastone inflammatory condition. Two or more therapies, including one or moreCD69 antagonist, can be administered in synergistic amounts.Accordingly, the administration of two or more therapies can have asynergistic effect on the decrease in one or more symptoms of colitisand/or hepatitis, or other inflammatory condition, whether administeredsimultaneously, sequentially, or in any combination. A first therapy canincrease the efficacy of a second therapy greater than if second therapywas employed alone, or a second therapy increases the efficacy of afirst therapy, or both. The effect of administering two or moretherapies can be such that the effect on decreasing one or more symptomof colitis and/or hepatitis is greater than the additive effect of eachbeing administered alone. When given in synergistic amounts, one therapycan enhance the efficacy of one or more other therapy on the decrease inone or more symptoms of colitis and/or hepatitis, even if the amount ofone or more therapy alone would have no substantial effect on one ormore symptom of colitis and/or hepatitis. Measurements and calculationsof synergism can be performed as described in Teicher, “Assays for InVitro and In Vivo Synergy,” in Methods in Molecular Medicine, vol. 85:Novel Anticancer Drug Protocols, pp. 297-321 (2003) and/or bycalculating the combination index (CI) using CalcuSyn software.

Exact formulation, route of administration and dosage can be chosen bythe individual physician in view of the patient's condition. [See, e.g.,Fingl et. al., in The Pharmacological Basis of Therapeutics, 1975, Ch. 1p. I.] The attending physician can determine when to terminate,interrupt, or adjust administration due to toxicity, or to organdysfunctions. Conversely, the attending physician can also adjusttreatment to higher levels if the clinical response were not adequate,precluding toxicity. The magnitude of an administrated dose in themanagement of disorder of interest will vary with the severity of thedisorder to be treated and the route of administration. The severity ofthe disorder can, for example, be evaluated, in part, by standardprognostic evaluation methods. The dose and dose frequency, can varyaccording to the age, body weight, and response of the individualpatient. A program comparable to that discussed above can be used inveterinary medicine.

Use of pharmaceutically acceptable carriers to formulate the compoundsherein disclosed for the practice of the invention into dosages suitablefor systemic administration is within the scope of the presentinvention. With proper choice of carrier and suitable manufacturingpractice, the compositions relevant to the present invention, inparticular, those formulated as solutions, can be administeredparenterally, such as by intravenous injection. The compounds can beformulated readily using pharmaceutically acceptable carriers well knownin the art into dosages suitable for oral administration. Such carriersenable the compounds relevant to the present invention to be formulatedas tablets, pills, capsules, liquids, gels, syrups, slurries, tablets,dragees, solutions, suspensions and the like, for oral ingestion by apatient to be treated.

The therapeutic agent can be prepared in a depot form to allow forrelease into the body to which it is administered is controlled withrespect to time and location within the body (see, for example, U.S.Pat. No. 4,450,150). Depot forms of therapeutic agents can be, forexample, an implantable composition containing the therapeutic agent anda porous or non-porous material, such as a polymer, wherein thetherapeutic agent is encapsulated by or diffused throughout the materialand/or degradation of the non-porous material. The depot is thenimplanted into the desired location within the body and the therapeuticagent is released from the implant at a predetermined rate.

The therapeutic agent that is used in the present invention can beformed as a composition, such as a pharmaceutical composition containinga carrier and a therapeutic compound. Pharmaceutical compositionscontaining the therapeutic agent can include more than one therapeuticagent. The pharmaceutical composition can alternatively contain atherapeutic agent in combination with other pharmaceutically activeagents or drugs.

The carrier can be any suitable carrier. For example, the carrier can bea pharmaceutically acceptable carrier. With respect to pharmaceuticalcompositions, the carrier can be any of those conventionally used withconsideration of chemico-physical considerations, such as solubility andlack of reactivity with the active compound(s), and by the route ofadministration. In addition to, or in the alternative to, the followingdescribed pharmaceutical compositions, the therapeutic compounds of thepresent inventive methods can be formulated as inclusion complexes, suchas cyclodextrin inclusion complexes, or liposomes.

The pharmaceutically acceptable carriers described herein, for example,vehicles, adjuvants, excipients, and diluents; are well-known to thoseskilled in the art and are readily available to the public. Thepharmaceutically acceptable carrier can be chemically inert to theactive agent(s) and one which has no detrimental side effects ortoxicity under the conditions of use. The choice of carrier can bedetermined in part by the particular therapeutic agent, as well as bythe particular method used to administer the therapeutic compound. Thereare a variety of suitable formulations of the pharmaceutical compositionof the present invention. The following formulations for oral, aerosol,parenteral, subcutaneous, transdermal, transmucosal, intestinal,intramedullary injections, direct intraventricular, intravenous,intranasal, intraocular, intramuscular, intraarterial, intrathecal,intraperitoneal, rectal, and vaginal administration are exemplary andare in no way limiting. More than one route can be used to administerthe therapeutic agent, and in some instances, a particular route canprovide a more immediate and more effective response than another route.Depending on the specific disorder being treated, such agents can beformulated and administered systemically or locally. Techniques forformulation and administration may be found in Remington'sPharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, Pa.(1990).

Formulations suitable for oral administration can include (a) liquidsolutions, such as an effective amount of the inhibitor dissolved indiluents, such as water, saline, or orange juice; (b) capsules, sachets,tablets, lozenges, and troches, each containing a predetermined amountof the active ingredient, as solids or granules; (c) powders; (d)suspensions in an appropriate liquid; and (e) suitable emulsions. Liquidformulations can include diluents, such as water and alcohols, forexample, ethanol, benzyl alcohol, and the polyethylene alcohols, eitherwith or without the addition of a pharmaceutically acceptablesurfactant. Capsule forms can be of the ordinary hard or soft shelledgelatin type containing, for example, surfactants, lubricants, and inertfillers, such as lactose, sucrose, calcium phosphate, and corn starch.Tablet forms can include one or more of lactose, sucrose, mannitol, cornstarch, potato starch, alginic acid, microcrystalline cellulose, acacia,gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium,talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid,and other excipients, colorants, diluents, buffering agents,disintegrating agents, moistening agents, preservatives, flavoringagents, and other pharmacologically compatible excipients. Lozenge formscan contain the inhibitor in a flavor, usually sucrose and acacia ortragacanth, as well as pastilles containing the inhibitor in an inertbase, such as gelatin and glycerin, or sucrose and acacia, emulsions,gels, and the like containing, in addition to, such excipients as areknown in the art.

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers can be added.

The therapeutic agent, alone or in combination with other suitablecomponents, can be made into aerosol formulations to be administered viainhalation. These aerosol formulations can be placed into pressurizedacceptable propellants, such as dichlorodifluoromethane, propane,nitrogen, and the like. They also can be formulated as pharmaceuticalsfor non-pressurized preparations, such as in a nebulizer or an atomizer.Such spray formulations also may be used to spray mucosa. Topicalformulations are well known to those of skill in the art. Suchformulations are particularly suitable in the context of the inventionfor application to the skin.

Injectable formulations are in accordance with the present invention.The parameters for effective pharmaceutical carriers for injectablecompositions are well-known to those of ordinary skill in the art [see,e.g., Pharmaceutics and Pharmacy Practice, J. B. Lippincott Company,Philadelphia, Pa., Banker and Chalmers, eds., pages 238250 (1982), andASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622 630(1986)]. For injection, the agents of the present invention can beformulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks's solution, Ringer's solution, orphysiological saline buffer. For such transmucosal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art.

Formulations suitable for parenteral administration can include aqueousand non-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The therapeutic agent can be administered in a physiologicallyacceptable diluent in a pharmaceutical carrier, such as a sterile liquidor mixture of liquids, including water, saline, aqueous dextrose andrelated sugar solutions, an alcohol, such as ethanol or hexadecylalcohol, a glycol, such as propylene glycol or polyethylene glycol,poly(ethyleneglycol) 400, glycerol, dimethylsulfoxide, ketals such as2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, oils, fatty acids, fattyacid esters or glycerides, or acetylated fatty acid glycerides with orwithout the addition of a pharmaceutically acceptable surfactant, suchas a soap or a detergent, suspending agent, such as pectin, carbomers,methylcellulose, hydroxypropylmethylcellulose, orcarboxymethylcellulose, or emulsifying agents and other pharmaceuticaladjuvants.

Oils, which can be used in parenteral formulations, include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includeoleic acid, stearic acid, and isostearic acid. Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters.

Suitable soaps for use in parenteral formulations include fatty alkalimetal, ammonium, and triethanolamine salts, and suitable detergentsinclude (a) cationic detergents such as, for example, dimethyl dialkylammonium halides, and alkyl pyridinium halides, (b) anionic detergentssuch as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin,ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionicdetergents such as, for example, fatty amine oxides, fatty acidalkanolamides, and polyoxyethylenepolypropylene copolymers, (d)amphoteric detergents such as, for example, alkyl-β-aminopropionates,and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixturesthereof.

The parenteral formulations can contain from about 0.5% to about 25% byweight of the drug in solution. Preservatives and buffers can be used.In order to minimize or eliminate irritation at the site of injection,such compositions may contain one or more nonionic surfactants having ahydrophilic-lipophilic balance (HLB) of from about 12 to about 17. Thequantity of surfactant in such formulations will typically range fromabout 5% to about 15% by weight. Suitable surfactants includepolyethylene glycol sorbitan fatty acid esters, such as sorbitanmonooleate and the high molecular weight adducts of ethylene oxide witha hydrophobic base, formed by the condensation of propylene oxide withpropylene glycol. The parenteral formulations can be presented inunit-dose or multi-dose sealed containers, such as ampoules and vials,and can be stored in a freeze-dried (lyophilized) condition requiringonly the addition of the sterile liquid excipient, for example, water,for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions can be prepared from sterile powders,granules, and tablets of the kind previously described.

The therapeutic agent can be made into suppositories by mixing with avariety of bases, such as emulsifying bases or water-soluble bases.Formulations suitable for vaginal administration can be presented aspessaries, tampons, creams, gels, pastes, foams, or spray formulascontaining, in addition to the active ingredient, such carriers as areknown in the art to be appropriate.

Agents intended to be administered intracellularly may be administeredusing techniques well known to those of ordinary skill in the art. Forexample, such agents can be encapsulated into liposomes. Liposomes arespherical lipid bilayers with aqueous interiors. Molecules present in anaqueous solution at the time of liposome formation are incorporated intothe aqueous interior. The liposomal contents are both protected from theexternal microenvironment and, because liposomes fuse with cellmembranes, are efficiently delivered into the cell cytoplasm.Additionally, due to their hydrophobicity, small organic molecules maybe directly administered intra-cellularly. Materials and methodsdescribed for one aspect of the present invention can also be employedin other aspects of the present invention. For example, a material sucha nucleic acid or antibody described for use in screening assays canalso be employed as therapeutic agents and vice versa.

The present invention includes the followingaspects/embodiments/features in any order and/or in any combination:

1. The present invention relates to a method of treating or reducing atleast one inflammatory condition and/or the susceptibility to at leastone inflammatory condition comprising:

administering at least one CD69 antagonist to a subject, wherein thesubject has been diagnosed with at least one inflammatory condition, orsusceptibility to the same, comprising colitis, hepatitis, asusceptibility to colitis, a susceptibility to hepatitis, or anycombination thereof, in an amount effective to treat or reduce theinflammatory condition and/or the susceptibility to the at least oneinflammatory condition.

2. The method of any preceding or following embodiment/feature/aspect,wherein the condition comprises colitis.3. The method of any preceding or following embodiment/feature/aspect,further comprising:

monitoring at least one symptom of colitis.

4. The method of any preceding or following embodiment/feature/aspect,wherein the at least one symptom comprises at least one of epithelialcell lesions, acute inflammation, diarrhea, rectal bleeding, and/orweight loss.5. The method of any preceding or following embodiment/feature/aspect,further comprising:

continuing administration if the at least one symptom persists, fallswithin a given range, or remains above or below a threshold value; or

discontinuing administration if the at least one symptom fails topersist, fails to fall within a given range, or no longer remains aboveor below a threshold value.

6. The method of any preceding or following embodiment/feature/aspect,wherein the symptom comprises at least one of epithelial cell lesions,acute inflammation, diarrhea, rectal bleeding, and weight loss.7. The method of any preceding or following embodiment/feature/aspect,wherein the condition comprises hepatitis.8. The method of any preceding or following embodiment/feature/aspect,further comprising:

monitoring at least one symptom of hepatitis.

9. The method of any preceding or following embodiment/feature/aspect,wherein the symptom comprises at least one of acute inflammation, raisedlevel of aspartate aminotransferase (AST), and raised level of alanineaminotransferase (ALT).10. The method of any preceding or following embodiment/feature/aspect,further comprising:

continuing administration if the at least one symptom persists, fallswithin a given range, or remains above or below a threshold value; or

discontinuing administration if the at least one symptom fails topersist, fails to fall within a given range, or no longer remains aboveor below a threshold value.

11. The method of any preceding or following embodiment/feature/aspect,wherein the symptom comprises at least one of acute inflammation, raisedlevel of aspartate aminotransferase (AST), and/or raised level ofalanine aminotransferase (ALT).12. The method of any preceding or following embodiment/feature/aspect,wherein the CD69 antagonist administered comprises an antibody or apolypeptide comprising an antigen-binding fragment of the antibody.13. The method of any preceding or following embodiment/feature/aspect,wherein the antibody is a chimeric antibody.14. The method of any preceding or following embodiment/feature/aspect,wherein the antibody is a humanized antibody.15. The method of any preceding or following embodiment/feature/aspect,wherein the antibody is a completely humanized antibody.16. The method of any preceding or following embodiment/feature/aspect,wherein the antibody is a monoclonal antibody.17. The method of any preceding or following embodiment/feature/aspect,wherein the antibody is a polyclonal antibody.18. The method of any preceding or following embodiment/feature/aspect,wherein the antibody is a single-chain antibody.19. The method of any preceding or following embodiment/feature/aspect,wherein the antibody or polypeptide is conjugated to a functional agentto form an immunoconguate.20. The method of any preceding or following embodiment/feature/aspect,wherein the functional agent is a cytotoxic agent that is an antibiotic,a radioactive isotope, a nucleolytic enzyme, a toxin, or any combinationthereof.21. The method of any preceding or following embodiment/feature/aspect,wherein the antibody or the polypeptide, comprising an antigen-bindingfragment of the antibody, binds to an extracellular domain of CD69.22. The method of any preceding or following embodiment/feature/aspect,wherein the CD69 antagonist comprises an aptamer that binds CD69.23. The method of any preceding or following embodiment/feature/aspect,wherein the aptamer comprises a nucleic acid.24. The method of any preceding or following embodiment/feature/aspect,wherein the aptamer comprises RNA.25. The method of any preceding or following embodiment/feature/aspect,wherein the aptamer comprises DNA.26. The method of any preceding or following embodiment/feature/aspect,wherein the aptamer comprises an amino acid.27. The method of any preceding or following embodiment/feature/aspect,wherein the CD69 antagonist comprises a mRNA antagonist.28. The method of any preceding or following embodiment/feature/aspect,wherein the mRNA antagonist comprises RNA interference.29. The method of any preceding or following embodiment/feature/aspect,wherein the mRNA antagonist comprises a ribozyme.30. The method of any preceding or following embodiment/feature/aspect,wherein two or more CD69 antagonists are administered.31. The method of any preceding or following embodiment/feature/aspect,wherein the two or more CD69 antagonists act synergistically to treat orreduce the inflammatory condition and/or the susceptibility to the atleast one inflammatory condition.32. The method of any preceding or following embodiment/feature/aspect,wherein at least one CD69 antagonist is administered in combination withone or more additional therapies directed to colitis, hepatitis, asusceptibility to colitis, a susceptibility to hepatitis, or anycombination thereof.33. The method of any preceding or following embodiment/feature/aspect,wherein the combination of the at least one CD69 antagonist and the oneor more additional therapies act synergistically to treat or reduce thesusceptibility to the at least one inflammatory condition.34. The method of any preceding or following embodiment/feature/aspect,wherein the CD69 antagonist comprises an anti-CD69 antibody and the oneor more additional therapy comprises one or more anti-colitis therapythat is at least one anti-diarrheal medication, at least oneaminosalicylate, at least one corticosteroid, at least oneimmunosuppressant, or any combination thereof.35. The method of any preceding or following embodiment/feature/aspect,wherein the CD69 antagonist comprises an anti-CD69 antibody and the oneor more additional therapy comprises one or more anti-hepatitis Atherapy that is at least one vaccine, at least one immunoglobulin, orany combination thereof.36. The method of any preceding or following embodiment/feature/aspect,wherein the CD69 antagonist comprises an anti-CD69 antibody and the oneor more additional therapy comprises one or more anti-hepatitis Btherapy that is at least one vaccine, at least one interferon, at leastone nucleoside reverse transcriptase inhibitor, or any combinationthereof.37. The method of any preceding or following embodiment/feature/aspect,wherein the CD69 antagonist comprises an anti-CD69 antibody and the oneor more additional therapy comprises one or more anti-hepatitis Ctherapy that is at least one vaccine, at least one peginterferon,ribavirin, or any combination thereof.

The present invention can include any combination of these variousfeatures or embodiments above and/or below as set forth in sentencesand/or paragraphs. Any combination of disclosed features herein isconsidered part of the present invention and no limitation is intendedwith respect to combinable features.

The following examples are given to illustrate the nature of theinvention. It should be understood, however, that the present inventionis not to be limited to the specific conditions or details set forth inthese examples. Data can be expressed as mean with SEM. Disease activityindex can be statistically analyzed using the Mann-Whitney U test.Differences in parametric data can be evaluated by the Student's t test.Differences of p<0.05 are considered statistically significant unlessotherwise indicated. The variance of the groups can be tested forequality by F test prior to t test analysis. The role of CD69 isinvestigated using mouse models of colitis and hepatitis, and it isfound that CD69 plays a significant role in the induction of bothcolitis and hepatitis. Furthermore, administration of anti-CD69 mAb canresult in a dramatic reduction in the extent of colitis and hepatitis,establishing that CD69 mAb can be used for the treatment of, orreduction in susceptibility to, colitis and hepatitis, as well asrelated inflammatory conditions.

EXAMPLES Example 1

Attenuated DSS-induced colitis in CD69-deficient mice. The aim of thisstudy is to evaluate the role of CD69 in the development of DSS-inducedcolitis. DSS-induced colitis is achieved by adding 4% DSS to thedrinking water to CD69−/−, CD69+/−, and WT mice for 7 days.CD69-deficient (CD69-KO) mice (Murata et al., Int. Immunol., 15:987-992(2003)) are obtained by backcrossing with BALB/c or C57BL/6 15 times.BALB/c and C57BL/6 mice are purchased from Charles River Laboratories(Tokyo, Japan). All mice are maintained under specific-pathogen-freeconditions. All animal care was carried out in accordance with theguidelines of Chiba University and Yamaguchi University.

Colitis was induced in 7 to 8-week-old mice with 4% (w/v) DSS (MW36,000-50,000; MP Biomedicals, Aurora, Ohio) in the drinking water thatwas filter-purified (Millipore Corp., Bedford, Mass.) for 7 days. Fromday 7 onwards, the animals received normal drinking water. DSSconsumption, body weight, stool consistency and fecal blood loss wererecorded daily. Fecal blood loss was assessed using the Hemocult test(NACALAI TESQUE Inc., Kyoto, Japan). A disease activity index (DAI)(Cooper et al., Lab. Invest., 69:238-249 (1993)) was calculated asdescribed in Table 1. At day 8 or 20, the mice were sacrificed. Aftermeasuring colon length, one half of the colon was fixed in 10%(vol./vol.) formalin, paraffin embedded and stained with H&E forhistological examination. The other half was frozen in liquid nitrogenand used for cytokine measurements and RNA extraction.

TABLE 1 Scoring of Disease Activity Index Weight Stool Fecal Score Lossconsistency^(a) Blood 0 None Normal Normal 1  1-5% 2  5-10% Loose stoolsHemoccult+ 3 10-20% 4   >20% Diarrhea Gross bleeding The diseaseactivity index is the combined scores of weight loss, stool consistencyand bleeding divided by three. ^(a)Normal stools = well-formed pellets,loose stools = pasty and semi-formed stools which do not stick to theanus, diarrhea = liquid stools that stick to the anus.

Survival rates were significantly increased in CD69−/−, CD69+/− micecompared with those in WT mice after 4% DSS administration (FIG. 1A).Weight, stool consistency and blood loss were scored to calculate thedisease activity index (DAI) as described in Table 1. Severity ofclinical symptoms was significantly decreased in both CD69−/− andCD69+/− mice compared to WT mice (FIG. 1B). CD69−/− and CD69+/− micealso showed significant protection against DSS-induced colitis asindicated by the weight loss (FIG. 1C).

Another sign of disease activity noted in DSS-treated WT mice wascolonic shortening (FIGS. 2A, 2B). Macroscopic examinations on day 8after DSS administration revealed that shortening of colon length ofCD69KO mice was significantly inhibited compared with that of WT mice(FIGS. 2A, 2B). On histological examination, crypt damage, ulceration,and infiltration of inflammatory cells were observed in the colons ofDSS treated WT mice (FIG. 2C). On the other hand, histological analysisof colons from DSS treated CD69KO mice showed greatly reduced numbers ofinfiltrating cells and degree of mucosal injury (FIG. 2C). Theexpression of CD69 in the infiltrating inflammatory cells in the colonsof DSS treated WT mice was examined by immunohistochemistry (FIG. 2D).As shown in FIG. 2D, significant expression of CD69 was detected in theinfiltrating inflammatory cells in the colons. No significant expressionof CD69 was observed in the colons of untreated mice. These resultsindicate that DSS-induced colitis was attenuated in CD69-deficient mice.

Example 2

DSS-induced colitis was restored partially by cell transfer of wild-typeCD4 T cells into CD69-KO mice. To investigate the cellular basisunderlying the involvement of CD69 in the pathogenesis of DSS-inducedcolitis, cell transfer experiments were performed in which whole spleencells, CD4 T cells, or neutrophils from WT mice were adoptivelytransferred into CD69KO mice.

Splenic CD4+ T cells from wild-type (WT) BALB/c mice were purified usingCD4+ T cell isolation kit (Miltenyi Biotec K.K. of Tokyo) and Auto-MACSsorter (Miltenyi Biotec K.K.), yielding a purity of >98%. Whole spleencells or CD4+ T cells were administered intravenously through the tailvain to CD69-KO mice (3×10⁷ cells/mouse) on day −1. For neutrophilpreparation, wild-type BALB/c mice were injected intraperitoneally with2 ml of 4% thioglycolate (Merck, Darmstadt, Germany) and peritonealneutrophils were recovered 4 h later by collecting peritoneal lavagewith 5 ml of saline (Ajuebor et al., J. Immunol., 162:1685-1691 (1999)).Neutrophils in the peritoneal lavage were stained with biotin-conjugatedGr-1 and streptavidin-microbeads, then purified using Auto-MACS sorter,yielding a purity of >90%. Fifteen million neutrophils were injectedintravenously into CD69 KO mice on day 0 and 2.

As shown in FIG. 3A, survival rates were significantly decreased by thecell transfer of WT CD4 T cells in CD69KO mice. On the other hand, thedecrease of survival rates was not observed by cell transfer of wholespleen cells or neutrophils from WT mice. Severity of clinical symptomsas indicated by the disease activity index (DAI) was substantiallyrestored in CD69KO mice transferred CD4 T cells or whole spleen cellsfrom WT mice (FIG. 3B). No significant changes in the weight loss wereobserved by cell transfer of whole spleen cells, CD4 T cells orneutrophils from WT mice (FIG. 3C). On the other hand, DSS-inducedshortening of colon length was restored in CD69KO mice transferred wholespleen cells, CD4 T cells or neutrophils from WT mice (FIG. 3D).Histological analysis of colons from DSS treated CD69KO mice transferredCD4 T cells or whole spleen cells from WT mice showed substantialrestoration of the inflammatory cell infiltration and mucosal injury(FIG. 3E). These results indicated that CD69 expression on CD4 T cellsplayed a role in the induction of the DSS-induced colitis.

Example 3

Attenuation of DSS-induced colitis by anti-CD69 antibody administration.In order to explore the therapeutic effect of the administration ofanti-CD69 mAb during DSS-induced colitis, WT BALB/c mice were treatedwith anti-CD69 mAb or control antibody on day 0, and then survival ofeach group during DSS-induced colitis was recorded daily. For theanti-CD69 antibody treatment, mice were injected with anti-CD69 mAb(H1.2F3, 500 μg/mouse) intraperitoneally on day 0. The data waspresented as representative of at least three individual experiments.

Survival rates were significantly increased by in vivo anti-CD69treatment compared with those in control antibody-treated WT mice afterDSS administration (FIG. 4A). Severity of clinical symptoms indicated bythe disease activity index (DAI) was substantially protected byanti-CD69 mAb treatment (FIG. 4B). The weight loss was slightlyprotected by anti-CD69 mAb treatment and recovered in survived mice atday 20 (FIG. 4C). On the other hand, DSS-induced shortening of colonlength was not protected by anti-CD69 mAb treatment (FIG. 4D). Onhistological examination, crypt damage, ulceration, and infiltration ofinflammatory cells were greatly reduced by anti-CD69 mAb treatment (FIG.4E). Thus, a therapeutic effect of anti-CD69 antibody was indicated inthe DSS-induced colitis model.

Example 4

Cytokine, chemokine and chemokine receptor expression. This exampledemonstrates that the CD69-mediated protection observed in murine DSScolitis is reflected by changes in mRNA expression of cytokines andchemokines related to ulcerative colitis. Mice were sacrificed at day 8and the colon tissues were harvested. Whole colonic RNA was isolated,reverse transcribed into cDNA and the expressions of IL-1β, IL-6, IL-10,CCR2, CCR3, CCL2, CCL4 and CCL5 are determined by real-time quantitativePCR. The amounts of mRNAs are normalized by the HPRT signal in therespective samples. Whole colonic RNA was extracted using Trizol(Invitrogen). RNA concentration was determined spectrophotometricallyand quality was assessed after agarose electrophoresis. cDNA synthesisand Quantitative RT-PCR is performed (Nigo et al., Proc. Natl. Acad.Sci. U.S.A., 103:2286-2291 (2006)). The primers for Taq Man probes forthe detection of IL-1β, IL-6, IL-10, CCR2, CCR3, CCL2, CCL4, CCL5 andHPRT were purchased from Applied Biosystems. The expression wasnormalized by the HPRT signal.

After 8 days of DSS exposure, a significant increase in the expressionlevels of IL-1β, IL-6, CCR2, CCR3, CCL2, CCL4 and CCL5 mRNA was seen inthe WT mice (FIG. 5). However, mRNA expression levels of IL-1β, IL-6,CCR3, CCL2 and CCL4 were not increased in DSS treated CD69KO mice (FIG.5). Interestingly, mRNA expression level of IL-10 was significantlyincreased in DSS treated CD69KO mice compared with that in DSS treatedWT mice.

Example 5

Attenuated ConA-induced hepatitis in CD69-deficient mice. Thephysiological roles of CD69 in ConA-induced hepatitis usingCD69-deficient mice was examined. Con A (Sigma) was dissolved inpyrogen-free PBS and intravenously injected into C57BL/6 mice throughthe tail vein at a dose of 10 mg/kg. Sera from individual mice wereobtained 12 h after ConA injection. Serum aminotransferase [alanineaminotransferase (ALT) and aspartate aminotransferase (AST)] activitieswas measured by the standard photometric method using an automaticanalyzer (Fuji Film Medical, Tokyo, Japan).

The hepatitis was evaluated by measuring the levels of aspartateaminotransferase (AST) and alanine aminotransferase (ALT) in plasma 12 hafter ConA injection (10 mg/ml). In the ConA-induced hepatitis model,the levels of AST and ALT increased 4 h after ConA injection, andthereafter increased rapidly reaching the peak values at 12 h timepoint. The levels of AST and ALT were decreased in CD69-deficient micein comparison to WT mice (FIG. 6A). A histological analysis of the liverwas also performed. After ConA-injection, liver damage accompanied withincreased apoptotic hepatocytes was apparently attenuated inCD69-deficient mice as compared to that observed in WT mice (FIG. 6B).

Example 6

Attenuation of ConA-induced hepatitis by anti-CD69 antibodyadministration. The administration of anti-CD69 mAb was examined todetermine whether such administration inhibits liver injury induced byintravenous injection of ConA. For the anti-CD69 antibody treatment,mice were injected with anti-CD69 mAb (H1.2F3, 400 μg/mouse)intraperitoneally 30 minutes before the injection of ConA. Forhistological analysis, the livers from individual mice were fixed in 10%formalin, embedded in paraffin, sectioned, and stained with H&E forhistological examination. Specimens are examined under a lightmicroscope.

The administration of anti-CD69 mAb (400 μg/mouse) before the injectionof ConA significantly suppressed the increased levels of AST and ALT.The histological changes in the liver 12 h after ConA injection werealso evaluated. Histological examination of the liver by H&E stainingrevealed focal or massive severe necrosis in the area between thecentral veins and the portal tracts of ConA-injected mice (FIG. 7B).Pre-administration of anti-CD69 mAb clearly suppressed the severenecrosis and apoptosis in the liver (FIG. 7B). The treatment of normalmice with anti-CD69 mAb alone had no effects on the levels of AST andALT. Thus, a therapeutic effect of anti-CD69 antibody was indicated inthe ConA-induced hepatitis model. IFN-gamma producing CD4 T cells andNKT cells also appeared to be good therapeutic target cells inConA-induced hepatitis.

Applicants specifically incorporate the entire contents of all citedreferences in this disclosure. Further, when an amount, concentration,or other value or parameter is given as either a range, preferred range,or a list of upper preferable values and lower preferable values, thisis to be understood as specifically disclosing all ranges formed fromany pair of any upper range limit or preferred value and any lower rangelimit or preferred value, regardless of whether ranges are separatelydisclosed. Where a range of numerical values is recited herein, unlessotherwise stated, the range is intended to include the endpointsthereof, and all integers and fractions within the range. It is notintended that the scope of the invention be limited to the specificvalues recited when defining a range.

Other embodiments of the present invention will be apparent to thoseskilled in the art from consideration of the present specification andpractice of the present invention disclosed herein. It is intended thatthe present specification and examples be considered as exemplary onlywith a true scope and spirit of the invention being indicated by thefollowing claims and equivalents thereof.

1. A method of treating or reducing at least one inflammatory conditionor the susceptibility to at least one inflammatory condition comprising:administering at least one CD69 antagonist to a subject, wherein thesubject has been diagnosed with at least one inflammatory condition, orsusceptibility to the same, comprising colitis, hepatitis, asusceptibility to colitis, a susceptibility to hepatitis, or anycombination thereof, in an amount effective to treat or reduce the atleast one inflammatory condition or the susceptibility to the at leastone inflammatory condition.
 2. The method of claim 1, wherein thecondition comprises colitis.
 3. The method of claim 2, furthercomprising: monitoring at least one symptom of colitis.
 4. The method ofclaim 3, wherein the at least one symptom comprises epithelial celllesions, acute inflammation, diarrhea, rectal bleeding, weight loss, orany combinations thereof.
 5. The method of claim 3, further comprising:continuing administration if the at least one symptom persists, fallswithin a given range, or remains above or below a threshold value; ordiscontinuing administration if the at least one symptom fails topersist, fails to fall within a given range, or no longer remains aboveor below a threshold value.
 6. The method of claim 4, wherein thesymptom comprises epithelial cell lesions, acute inflammation, diarrhea,rectal bleeding, weight loss, or any combinations thereof.
 7. The methodof claim 1, wherein the condition comprises hepatitis.
 8. The method ofclaim 7, further comprising: monitoring at least one symptom ofhepatitis.
 9. The method of claim 8, wherein the symptom comprises acuteinflammation, raised level of aspartate aminotransferase (AST), raisedlevel of alanine aminotransferase (ALT), or any combinations thereof.10. The method of claim 8, further comprising: continuing administrationif the at least one symptom persists, falls within a given range, orremains above or below a threshold value; or discontinuingadministration if the at least one symptom fails to persist, fails tofall within a given range, or no longer remains above or below athreshold value.
 11. The method of claim 4, wherein the symptomcomprises acute inflammation, raised level of aspartate aminotransferase(AST), raised level of alanine aminotransferase (ALT), or anycombinations thereof.
 12. The method of claim 1, wherein the CD69antagonist administered comprises an antibody or a polypeptidecomprising an antigen-binding fragment of the antibody.
 13. The methodof claim 12, wherein the antibody is a chimeric antibody.
 14. The methodof claim 12, wherein the antibody is a humanized antibody.
 15. Themethod of claim 12, wherein the antibody is a completely humanizedantibody.
 16. The method of claim 12, wherein the antibody is amonoclonal antibody.
 17. The method of claim 12, wherein the antibody isa polyclonal antibody.
 18. The method of claim 12, wherein the antibodyis a single-chain antibody.
 19. The method of claim 12, wherein theantibody or polypeptide is conjugated to a functional agent to form animmunoconguate.
 20. The method of claim 19, wherein the functional agentis a cytotoxic agent that is an antibiotic, a radioactive isotope, anucleolytic enzyme, a toxin, or any combination thereof.
 21. The methodof claim 12, wherein the antibody or the polypeptide, comprising anantigen-binding fragment of the antibody, binds to an extracellulardomain of CD69.
 22. The method of claim 1, wherein the CD69 antagonistcomprises an aptamer that binds CD69.
 23. The method of claim 22,wherein the aptamer comprises a nucleic acid.
 24. The method of claim23, wherein the aptamer comprises RNA.
 25. The method of claim 23,wherein the aptamer comprises DNA.
 26. The method of claim 22, whereinthe aptamer comprises an amino acid.
 27. The method of claim 1, whereinthe CD69 antagonist comprises a mRNA antagonist.
 28. The method of claim27, wherein the mRNA antagonist comprises RNA interference.
 29. Themethod of claim 27, wherein the mRNA antagonist comprises a ribozyme.30. The method of claim 1, wherein two or more CD69 antagonists areadministered.
 31. The method of claim 30, wherein the two or more CD69antagonists act synergistically to treat or reduce the susceptibility tothe at least one inflammatory condition.
 32. The method of claim 1,wherein at least one CD69 antagonist is administered in combination withone or more additional therapies directed to colitis, hepatitis, asusceptibility to colitis, a susceptibility to hepatitis, or anycombination thereof.
 33. The method of claim 32, wherein the combinationof the at least one CD69 antagonist and the one or more additionaltherapies act synergistically to treat or reduce the susceptibility tothe at least one inflammatory condition.
 34. The method of claim 32,wherein the CD69 antagonist comprises an anti-CD69 antibody and the oneor more additional therapy comprises one or more anti-colitis therapiesthat is at least one anti-diarrheal medication, at least oneaminosalicylate, at least one corticosteroid, at least oneimmunosuppressant, or any combination thereof.
 35. The method of claim32, wherein the CD69 antagonist comprises an anti-CD69 antibody and theone or more additional therapies comprises one or more anti-hepatitis Atherapy selected from at least one vaccine, at least one immunoglobulin,or any combination thereof.
 36. The method of claim 32, wherein the CD69antagonist comprises an anti-CD69 antibody and the one or moreadditional therapies comprises one or more anti-hepatitis B therapyselected from at least one vaccine, at least one interferon, at leastone nucleoside reverse transcriptase inhibitor, or any combinationthereof.
 37. The method of claim 32, wherein the CD69 antagonistcomprises an anti-CD69 antibody and the one or more additional therapiescomprises one or more anti-hepatitis C therapy selected from at leastone vaccine, at least one peginterferon, ribavirin, or any combinationthereof.